Surface expression vectors having pgsBCA the gene coding poly-gamma-glutamate synthetase, and a method for expression of target protein at the surface of microorganism using the vector

ABSTRACT

The present invention relates to a surface expression vector having pgsBCA, a gene coding poly-gamma-glutamate synthetase and a method for expression of target protein at the surface of microorganism using the vector. The vector, in which foreign genes are inserted, transforms microorganisms and makes foreign proteins expressed stably on the surface of microorganisms.

STATEMENT OF JOINT RESEARCH AGREEMENT

This invention was made as a result of activities undertaken within thescope of a joint research agreement between the following parties:Bioleaders Corp.; Korea Research Institute of Bioscience andBiotechnology; Chosun University; Genolac BL Corp.; Imbio Corp.; M.D.Lab Co., Ltd.; and Bioleaders Japan Corp.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. §371 andclaims the priority of International Patent Application No.PCT/KR02/01522 filed Aug. 9, 2002, which in turn claims priority ofKorean Patent Application No. 2001-48373 filed Aug. 10, 2001.

TECHNICAL FIELD

The current invention relates to novel expression vectors that canefficiently produce exogenous proteins on a microbial surface andexploit the cell outer membrane protein (pgsBCA) participating in thesynthesis of poly-γ-glutamate derived from a Bacillus sp. strain. Inaddition, the present invention relates to a method for expressing anexogenous protein on a microbial surface by exploiting the cell outermembrane protein (pgsBCA) participating in the synthesis ofpoly-γ-glutamate derived from a Bacillus sp. strain.

BACKGROUND ART

Recently, the use of surface expression to produce valuable exogenousproteins on cell surfaces has been attempted with bacteriophages,bacteria, and yeast for the purpose of creating new vaccines, screeningvarious kinds of antigens and antibodies, and fixing useful enzymes ontocell surfaces.

Originally, the idea of expressing exogenous proteins on a cell surfacewas to produce antigenic regions of peptides, especially for thelarge-scale stable expression of vaccines. Currently, pathogenicbacteria are randomly mutated to produce vaccines and screened tocollect bacteria with consistent and stable titers. However,unfortunately, the enzymatic activity is invariably lost after oraladministration to humans and animals. Therefore, many studies have beenconducted to over come this problem. Normally, the cell surface proteinof a Gram-negative bacterium is adopted and its gene ligated with anantigenic protein gene, which is then introduced to proper host cells sothat fusion proteins are efficiently produced on the cell surface. Therecombinant protein prepared through this procedure can be an effectiveantigen as it is protruded onto the cell surface. In particular,Gram-negative bacteria have been reported as most suitable forproduction, as the lipopolysaccharides (LPS) in the cell outer membraneenhance the antigenicity of the proteins expressed on the cell surface.

To express exogenous proteins on a cell surface, the presence of asecretion signal is required within the primary sequence, since thispasses the biosynthesized cell proteins through the cell membrane.Besides, in Gram-negative bacteria, the recombinant protein must alsopass though the cell inner membrane and space between the cellmembranes, be inserted and attached to the cell outer membrane, andfinally stably protruded to the external side of the cell membrane.

Practically, there are certain proteins that include such a secretionsignal and targeting signal and are stably protruded onto the cellsurface, for example, cell surface proteins, specific enzymes, and toxinproteins. As such, if these secretion and targeting signals areassociated with a proper promoter, exogenous proteins can besuccessfully expressed onto a bacterial surface.

In general, the cell surface proteins adopted for the surface expressionof foreign proteins can be basically classified to 4 kinds, a cell outermembrane, lipoprotein, secretion protein, and cell surface organprotein. Until now, the surface proteins present in Gram-negativebacteria, for example, LamB, PhoE, and OmpA, have been mainly utilizedto produce useful foreign proteins. However, these proteins presentstructural restrictions as regards the size of the insertable proteins,which are inserted into the protruded loop on the cell surface. Sincethe C- and N-termini of the inserted exogenous protein should bestereochemically close, if they are distant, connected peptides can beligated to reduce the distance between the two termini.

Concretely, if LamB and PhoE are used to insert an exogenous polypeptideconsisting of more than 50˜60 amino acids, structural constraints areinvoked preventing the creation of a stable protein on the cell membrane(Charbit, et al., J. Immunol., 139: 1658-1664, 1987; Agterberg, et al.,Vaccine, 8: 85-91, 1990). Although OmpA can be utilized to introduceexogenous proteins into the protruded loop, only a partial fragment ofOmpA containing a minimal targeting signal can actually be added due tothe structural constraint. β-lactamase has been expressed on a cellsurface by connecting the OmpA targeting signal at the C-terminus.

Recently, the ice-nucleation protein (INP) derived from Pseudomonas sp.was found to be a cell outer membrane of Gram-negative bacteria andutilized for surface expression (Jung et al., Nat. Biotechnol., 16:576-580, 1998; Jung et al., Enzyme Microb. Technol., 22(5): 348-354,1998; Lee et al., Nat. Biotechnol., 18: 645-648, 2000). Jung andcolleagues expressed levansucrase onto a cell surface using the icenucleation protein, consisting of the N-terminus, central repetitiveregion, and C-terminus, and ligating the levansucrase gene at theC-terminus, while also expressing carboxymethylcellulase using the icenucleation protein, consisting of the N-terminus, deleted centralrepetitive region, and C-terminus, and fusing the gene at theC-terminus, so as to assay the respective enzymatic activities. Inaddition, Lee and colleagues used the ice-nucleation protein, comprisingof just the N-terminus or the N-terminus and C-terminus, ligated withthe hepatitis B virus surface antigen and hepatitis C virus core antigenat each terminus, for expression on the cell surface of an Escherichiacoli or Salmonella typhi Ty21a strain, then confirmed that theseproteins were effective for complex live vaccines.

Lipoproteins have also been utilized as a surface protein for surfaceexpression. In particular, E. coli lipoproteins can pass through thecell inner membrane based on the secretion signal at the N-terminus andcontain L-cystein at the terminus directly connected to the cell outermembrane or inner membrane. A major lipoprotein, Lpp, is associated withthe cell outer membrane at the N-terminus and with peptidoglycan (PG) atthe C-terminus. Thus, if Lpp is connected with the OmpA fragment of thecell outer membrane protein, exogenous proteins can be stably expressedonto the cell surface of the cell outer membrane (Francisco, et al.,Proc. Natl. Acad. Sci. USA, 89: 2713-2717, 1992). This characteristichas also been used with another lipoprotein, TraT, to express foreignpeptides, such as the C3 epitope of the poliovirus, onto a cell surface(Felici, et al., J. Mol. Biol., 222: 301-310, 1991). Furthermore, thepeptidoglycan-associated lipoprotein (PAL), although not yet elucidatedas regards its precise function, has been adopted to produce recombinantantigens through surface expression (Fuchs, et al., Bio/Technology, 9:1369-1372, 1991). In this case, the C-terminus of PAL is ligated to thecell wall and the N-terminus to the recombinant antibody so as toexpress a fusion protein on the cell surface.

Meanwhile, even though secretion proteins that can pass through the cellouter membrane can be used as a surface protein, this has not beendeveloped in Gram-negative bacteria and only a few kinds of secretionproteins can help passage through the cell outer membrane in thepresence of specific proteins participating in the secretion mechanism.For example, Klebsiella sp. pullulanase as a lipoprotein is completelysecreted into a cell culture medium after its N-terminus is substitutedwith a lipid substance and attached to the cell outer membrane.Kornacker and colleagues expressed β-lactamase onto a cell surface whenusing the N-terminus fragment of pullulanase, yet the resulting fusionprotein of pullulanase-β-lactamase was instantly attached onto the cellsurface, then unfortunately separated into the cell culture medium. Inaddition, this process has also been exploited to produce alkalinephosphatase, a periplasmic space protein, yet the recombinant protein isnot stably expressed as at least 14 proteins are required for thesecretion (Kornacker, et al., Mol. Microbiol., 4: 1101-1109, 1990).

Moreover, IgA protease, derived from the pathogenic microbe Neisseriasp., has a specific secretion system with a fragment signal present atthe C-terminus, which makes the protease present at the N-terminusstably attached to the cell outer membrane. Once arriving at the cellouter membrane and protruding on the cell surface, the protease issecreted into the cell culture medium based on its hydrolytic capacity.Klauser and colleagues inconsistently expressed the B subunit of thecholera toxin with a molecular weight of about 12 kDa onto a cellsurface using this IgA protease fragment (Klauser, et al., EMBO J., 9:1991-1999, 1990). However, the secretion of the fused protein wasinhibited by the protein folding induced in the cell membrane spaceduring the secretion process.

Besides, in the case of Gram-negative bacteria, the cell suborganspresent on the cell surface and applicable for surface expression arecomposed of flagella, pili, and fimbriae etc. In detail, the B subunitof the cholera toxin and peptides derived from the hepatitis B virushave been consistently produced using flagellin as a subunit composed offlagella and identified as strongly binding with their antibodies(Newton, et al., Science, 244:70-72, 1989). Then, fimbrin, a subunitconstituting of threadlike fimbriae on the cell surface, has beenutilized to express exogenous peptides, yet only small peptides havebeen successfully produced (Hedegaard, et al., Gene, 85: 115-124, 1989).

Although the surface proteins of Gram-negative bacteria have alreadybeen used to perform surface expression, recently, the surface proteinsof Gram-positive bacteria have also been used for surface expression(Samuelson, et al., J. Bacteriol., 177: 1470-1476, 1995). Yet, even inthis case, a secretion signal for passing through the cell innermembrane and carrier for surface expression and attaching onto the cellmembrane are also needed. In fact, the secretion signal of the lipasederived from Staphylococcus hyicus and membrane attachment carrier ofprotein A derived from Staphylococcus aureus have been utilized toproduce a malaria blood stage antigen composed of 80 amino acids andalbumin attachment protein derived from Streptococcus protein G andefficiently express the resulting proteins onto the cell surface.

As described above, since much research has already focused on surfaceexpression with Gram-negative bacteria and Gram-positive bacteria, anumber of expression systems have already been developed for theproduction of valuable proteins and submitted for patent applications,especially in the USA, Europe, and Japan. In detail, 5 patent cases havedisclosed the use of the cell outer membrane proteins of Gram negativebacteria (WO 9504069, WO 9324636, WO 9310214, EP 603672, U.S. Pat. No.5356797), one patent application has reported the use of pili as a cellsurface organelle (WO 9410330), and one case using a cell surfacelipoprotein (WO 9504079).

As stated above, to express exogenous proteins onto a cell surface usinga cell outer membrane protein, the proper cell inner membrane andexogenous protein must be connected on a gene level, induced forbiosynthesis, and sustained on the cell outer membrane after passingstably through the cell inner membrane. To accomplish this procedure, acell inner membrane satisfying the following requirements should beselected, then applied to the carrier for surface expression: above all,the presence of a secretion signal for passing through the cell innermembrane, second, the presence of a targeting signal for stableattachment to the cell outer membrane, third, massive expression ontothe cell surface, and fourth, stable expression of the protein,regardless of its size.

However, carriers for surface expression that meet all theserequirements have not yet been developed. Currently, only the followingdisadvantages have been remedied.

Based on such a background, the present inventors investigated theapplication of a poly-γ-glutamate synthase gene (pgsBCA) derived from aBacillus sp. strain as a novel carrier for surface expression. As aresult, a novel expression vent or pgsBCA-containing gene that canefficiently produce exogenous proteins onto microbial surfaces wasdeveloped along with a method for successfully expressing exogenousproteins onto microbial surfaces on a large scale.

DISCLOSURE OF INVENTION

The object of the current invention is to provide a method for producingexogenous proteins on a microbial surface.

In detail, in the current invention, a new surface expression carrierthat can express foreign proteins onto the surfaces of Gram-negative andGram-positive microbes on a large scale was selected from the cell outermembrane proteins participating in the synthesis of poly-γ-glutamatefrom a Bacillus sp. strain. Then, utilizing this gene, a surfaceexpression vector that can express exogenous proteins or peptides ontomicrobial surfaces was constructed and transformed into various kinds ofhost cell in order to collect cell transformants for surface expression.

To accomplish the objectives of the present invention, a surfaceexpression vector is presented that contains one or more genes encodinga poly-γ-glutamate synthetase complex selected from among pgsb, pgsC,and pgsA.

In detail, the present invention presents a surface expression vectorfor producing proteins on a microbial surface, in which the pgsB, pgsc,and pgsA genes contain nucleotide sequences that are 80% homologous tothose of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3, respectively. Inaddition, a surface expression vector is also presented for producingproteins on a microbial surface that contain a gene encoding a targetprotein and transcription termination codon at the C-terminus.

Furthermore, a cell transformant is presented that is transformed usingthe above expression vector.

Finally, the current invention provides a method for expressing a targetprotein on a microbial surface of Gram-negative or Gram-positive hostcells based on the following steps:

(a) constructing a recombinant expression vector by inserting a geneencoding the target protein into the surface expression vector;

(b) transforming a Gram-negative host cell using the recombinant vector;and

(c) cultivating the transformed host cell and expressing the targetprotein on the surface of the host cell.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objective, features, and advantages of the presentinvention will be more clearly understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich;

FIG. 1 depicts the restriction maps of the surface expression vectorpGNBCA and recombinant expression vector pGNBCA-HB168, which useGram-negative bacteria as the host cell in the present invention.

FIG. 2 depicts the surface expression of the hepatitis B virus surfaceantigen protein in a Gram negative bacterium transformed with therecombinant expression vector pGNBCA-HB168 of the current inventionbased on performing Western blotting and fluorescence-activated cellsorting assays.

FIG. 3 depicts the restriction maps of the surface expression vectorpGNCA and recombinant expression vector pGNCA-HB168 of the presentinvention.

FIG. 4 depicts the surface expression of the hepatitis B virus surfaceantigen protein in a Gram-negative bacterium transformed with thesurface expression recombinant vectors pGNCA-HB168:A2, pGNCA-HB168:A3,and pGNHB-A:A4 of the present invention based on performing Westernblotting and fluorescence-activated cell sorting assays.

FIG. 5 depicts the restriction maps of the surface expression vectorpGNA and recombinant expression vector pGNA-HB168 of the currentinvention.

FIG. 6 depicts the restriction maps of the surface expression vectorpGNCA2 and recombinant expression vector pGNHB-A of the currentinvention.

FIG. 7 depicts the restriction maps of the surface expression vectorpGNC and recombinant expression vector pGNC-PreS1 of the currentinvention.

FIG. 8 depicts the surface expression pattern of the hepatitis B virussurface antigen PreS1 protein in a Gram negative bacterium transformedusing the surface expression recombinant vector pGNC-PreS1 of thecurrent invention based on performing a Western blotting assay.

FIG. 9 depicts the restriction maps of the surface expression vectorpHCE1LB:BCA and recombinant expression vector pHCE1LB:BCA-HB168 of thecurrent invention.

FIG. 10 depicts the surface expression pattern of the hepatitis B virussurface antigen determinant in a Gram-negative bacterium transformedusing the surface expression recombinant vector pHCE1LB:BCA-HB168 of thecurrent invention based on performing Western blotting andfluorescence-activated cell sorting assays.

FIG. 11 depicts the live vaccine efficacy of a Gram-negative bacteriumtransformed using the recombinant expression vector pHCE1LB:BCA-HB168 ofthe current invention.

FIG. 12 depicts the surface expression of the hepatitis B virus surfaceantigen determinant in a Gram-positive bacterium transformed using thesurface expression recombinant vector pHCE1LB:BCA-HB168 of the currentinvention based on performing Western blotting andfluorescence-activated cell sorting assays.

FIG. 13 depicts the live vaccine efficacy of a Gram-positive bacteriumtransformed using the recombinant expression vector pHCE1LB:BCA-HB168 ofthe current invention.

FIG. 14 depicts the restriction maps of the surface expression vectorpHCE1LB:A and recombinant expression vectors pHCE1LB:A-TGEN1 andpHCE1LB:A-PEDN of the current invention.

FIG. 15 depicts the surface expression pattern of the TGE virus Nprotein produced from Gram-negative (Escherichia coli) and Gram-positivebacteria transformed using the surface expression recombinant vectorpHCE1LB:A-TGEN1 of the current invention based on performing a Westernblotting assay.

FIG. 16 depicts the surface expression pattern of the PED virus Nprotein produced from Gram-negative and Gram-positive bacteriatransformed using the expression recombinant vector pHCE1LB:A-PEDN ofthe current invention based on performing a Western blotting assay.

FIG. 17 depicts the live vaccine efficacy of a Gram-positive bacteriumtransformed using the recombinant expression vectors pHCE1LB:A-TGEN1 andpHCE1LB:A-PEDN of the current invention.

FIG. 18 depicts the restriction maps of the surface expression vectorpHCE1LB:A and recombinant expression vectors pHCE1LB:A-PreS1 andpHCE1LB:A-PreS2 of the current invention.

FIG. 19 depicts the restriction maps of the surface expression vectorpHCE1LB:A and recombinant expression vectors pHCE1LB:A-PreS1:PreS2 andpHCE1LB:A-L of the current invention.

FIG. 20 depicts the surface expression pattern of the hepatitis B virusPreS1 and PreS1:PreS2 produced from a Gram-negative bacteriumtransformed using the expression recombinant vectors pHCE1LB:A-PreS1 andpHCE1LB:A-PreS1:PreS2, respectively, of the current invention andsurface expression pattern of the hepatitis B virus L protein producedfrom a Gram-negative bacterium transformed using the expressionrecombinant vector pHCE1LB:A-L of the current invention based onperforming a Western blotting assay.

FIG. 21 depicts the restriction maps of the surface expression vectorpHCE1LB:A-TNF-α of the current invention.

FIG. 22 depicts the restriction maps of the recombinant expressionvector pGNA-lipase of the current invention and lipase activityexpressed onto the cell surface of a Gram-negative bacterium transformedusing the recombinant expression vector pGNA-lipase.

FIG. 23 depicts the restriction maps of the recombinant expressionvector pGNA-amidase of the current invention and amidase activityexpressed onto the cell surface of a Gram-negative bacterium transformedusing the recombinant expression vector pGNA-amidase.

BEST MODE FOR CARRYING OUT PRESENT INVENTION

Hereinafter, the present invention will be described more clearly.

The protein encoded from the pgsBCA gene is a cell outer protein presentin Bacillus sp. strains and is polymer substance edible, soluble,anionic, biodegradable participating in the synthesis ofpoly-γ-glutamate, and produced from Bacillus subtilis (IF03336; Natto.Biochem. Biophys. Research Comm., 263, 6-12, 199, Bacillus licheniformis(ATCC 9945; Biotech. Bioeng., 4), 430-437, 1998), and Bacillus anthracis(J. Bacteriol., 171, 722-730, 1989) etc.

From the Natto strain (Bacillus subtilis IFO 3336), a cell membraneprotein (pgsBCA) was separated that was composed of a total of 922 aminoacids, precisely, 393 amino acids in pgsB, 149 amino acids in pgsC, and380 amino acid in pgsA. Ashiuchi et al. reported on the cloning of thepoly-γ-glutamate synthetase gene derived from the Bacillus natto strain,its transformation into Escherichia coli, and synthesis (Ashiuchi, etal., Biochem. Biophy. Research Comm, 263: 6-12, 1999).

However, the functions of the pgsBCA protein comprising thepoly-γ-glutamate synthetase complex have not yet been elucidated indetail. At least, pgsB is an amide ligase system among the proteinsmaking up the enzyme complex and interacts with the cell membrane orcell wall at a specific amino acid in the N-terminus of pgsB. pgsA has ahydrophilic amino acid sequence specific for the N-terminus andC-terminus that would appear to work as a secretion signal, targetingsignal, and attachment signal in association with pgsB for passingthrough the cell inner membrane.

The present inventors revealed that the cell outer membrane proteinparticipating in the synthesis of poly-γ-glutamate is advantageous as asurface expression carrier and can express exogenous proteins onto acell surface based on the structure and features of its primary aminoacid sequence. Concretely, there are various advantages. First, the cellouter membrane protein participating in the synthesis ofpoly-γ-glutamate can be expressed on a large scale for the synthesis ofpoly-γ-glutamate and extracellular secretion; second, the cell outermembrane protein expressed onto the cell surface participating in thesynthesis of poly-γ-glutamate can be maintained stably during the restperiod of the cell cycle; third, the cell outer membrane protein isstructurally protruded on the cell surface, especially in the case ofpgsA; fourth, the cell outer membrane protein (pgsBCA) participating inthe synthesis of poly-γ-glutamate originates from the cell surface of aGram-positive bacterium and can be expressed onto the cell surface ofeither a Gram-positive or Gram negative bacterium.

The present invention provides recombinant expression vectors that areuseful for expressing exogenous proteins on a cell surface by exploitingthe property of the cell outer membrane participating in the synthesisof poly-γ-glutamate. In detail, the surface expression vectors of thepresent invention are constructed to contain a secretion signal andtargeting signal in the primary sequence of the cell outer membraneprotein (pgsBCA) participating in the synthesis of poly-γ-glutamate.

Furthermore, the present invention provides a method for expressingexogenous proteins onto the microbial surface of both Gram-negative andGram-positive bacteria using the surface expression vector based onexploiting the features of the cell outer membrane protein participatingin the synthesis of poly-γ-glutamate. In detail, the method forpreparing exogenous proteins in the present invention can omit certainprocesses, such as cell sonication or protein purification, since theexogenous proteins are expressed onto the cell surface using the cellouter membrane protein participating in the synthesis ofpoly-γ-glutamate.

Therefore, the present invention provides various uses for exogenousproteins produced through the surface expression method. In detail, theproposed method is effective for producing antigens and antibodies,peptide libraries for screening antigens, attachment proteins oradsorption proteins, and physiologically active substances, etc.

In addition to the cell outer membrane protein participating in thesynthesis of poly-γ-glutamate derived from a Bacillus sp. strain, allkinds of surface expression vectors containing genes for the synthesisof poly-γ-glutamate can be included within the scope of the presentinvention.

Moreover, the surface expression vectors using the poly-γ-glutamatesynthetase gene in the present invention can be applied to all kinds ofmicrobial strains for surface expression. Preferably, these vectors canbe utilized for Gram-negative bacteria, especially Escherichia coli,Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacteriumbovis, and Shigella, and for Gram-positive bacteria, especiallyBacillus, Lactobacillus, Lactococcus, Staphylococcus, Lysteria,Monocytogenesis, and Streptococcus. All the methods used to manufactureexogenous proteins using these strains can be included within the scopeof the present invention.

Depending on the requirements, the poly-γ-glutamate synthetase gene canbe manipulated to insert various recognition sites for all or certainrestriction enzymes at the N-terminus or C-terminus. Hence, surfaceexpression vectors including these restriction enzyme recognition sitesare also within the scope of the present invention.

In detail, the current invention covers all the poly-γ-glutamatesynthetase genes derived from Bacillus sp. strains. Among these, thepgsA gene is used to insert the restriction enzyme recognition site atthe C-terminus and easily clone various kinds of exogenous proteingenes, thereby constructing the surface expression vector pGNBCA.

The current invention also presents the recombinant surface expressionvector pGNBCA-HB168, which can express an antigenic determinant forminga neutralizing antibody against an S antigen in a fused form onto thecell surface of a Gram negative bacterium. In detail, the cell outermembrane protein complex participating in the synthesis ofpoly-γ-glutamate is composed of pgsB, pgsC, and pgsA proteins, then theC-terminus of the pgsA protein gene is ligated with the N-terminus of anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen.

The current invention also presents the recombinant surface expressionvectors pGNCA-HB168, pGNA-HB168, and pGNHB-A, which can express anantigenic determinant forming a neutralizing antibody against an Santigen in a fused form onto the cell surface of a Gram negativebacterium. In detail, the cell outer membrane protein complexparticipating in the synthesis of poly-γ-glutamate is composed of eitherpgsC and pgsA proteins or only pgsA proteins, then the C-terminus of thepgsA protein gene, in the case of the former, or the N-terminus orC-terminus, in the case of the latter, is ligated with the N-terminus ofan antigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen.

The current invention also presents the recombinant surface expressionvector pGNC-PreS1, which can express an antigenic determinant forming aneutralizing antibody against an S antigen in a fused form on the cellsurface of a Gram negative bacterium. In detail, the cell outer membraneprotein complex participating in the synthesis of poly-γ-glutamate iscomposed of pgsC proteins, then the C-terminus of the pgsC protein geneis ligated with the N-terminus of the PreS1 antigen from among thehepatitis B virus surface antigens.

The current invention also presents the recombinant surface expressionvectors pHCE1LB:BCA and pHCE1LB:A, which modify the surface expressionvector pGNBCA for a Gram-negative bacterium and can be applied to bothGram-negative and Gram-positive bacteria. In detail, the cell outermembrane protein complex participating in the synthesis ofpoly-γ-glutamate is composed of either pgsB, pgsc, and pgsA proteins oronly pgsA proteins, then the C-terminus of the pgsA protein gene isligated with the exogenous protein gene.

The current invention also presents the recombinant surface expressionvector pHCE1LB:BCA-HB168, which can be applied to both Gram-negative andGram-positive bacteria and expresses an antigenic determinant forming aneutralizing antibody against an S antigen in a fused form on a cellsurface. In detail, the cell outer membrane protein complexparticipating in the synthesis of poly-γ-glutamate is composed of thepgsB, pgsc, and pgsA proteins, then the C-terminus of the pgsA proteingene is ligated with the N-terminus of an antigenic determinant forminga neutralizing antibody against the hepatitis B virus S antigen.

The current invention also presents the recombinant surface expressionvector pHCE1LB:A-TGEN1, which can be applied to both Gram-negative andGram-positive bacteria and expresses a nucleoprotein N protein onto acell surface in a fused form. In detail, the cell outer membrane proteincomplex participating in the synthesis of poly-γ-glutamate is composedof pgsA proteins, then the C-terminus of the pgsA protein gene isligated with the N-terminus of the partial nucleoprotein gene of theporcine transmissible gastric disease (TGE) virus.

The current invention also presents the recombinant surface expressionvector pHCE1LB:A-PEDN, which can be applied to both Gram-negative andGram-positive bacteria and expresses a nucleoprotein N protein onto acell surface in a fused form. In detail, the cell outer membrane proteincomplex participating in the synthesis of poly-γ-glutamate is composedof pgsA proteins, then the C-terminus of the pgsA protein gene isligated with the N-terminus of the nucleoprotein gene of the porcinediarrhea disease (PED) virus.

The current invention also presents the recombinant surface expressionvectors pHCE1LB:A-PreS1, pHCE1LB:A-PreS2, pHCE1LB:A-PreS1:PreS2, andpHCE1LB:A-L, which can be applied to both Gram-negative andGram-positive bacteria for the surface expression of exPreS1, PreS2,PreS1-PreS2 or total L protein among the hepatitis B virus surface L(PreS1-PreS2-S) proteins, respectively, in a fused form onto a cellsurface. In detail, the cell outer membrane protein complexparticipating in the synthesis of poly-γ-glutamate is composed of pgsAproteins, then the C-terminus of the pgsA protein gene is ligated withthe N-terminus of PreS1, PreS2, PreS1-PreS2, or the total L protein,respectively.

The current invention also presents the recombinant surface expressionvector pHCE1LB:A-TNF-α, which can be applied to both Gram-negative andGram-positive bacteria and expresses the TNF-α protein onto a cellsurface in a fused form. In detail, the cell outer membrane proteinparticipating in the synthesis of poly-γ-glutamate is composed of pgsAproteins, then the C-terminus pgsA protein gene is ligated with theN-terminus of the tumor necrosis factor α, a cytokine.

The current invention also presents the recombinant surface expressionvectors pGNA-lipase and pGNA-amydase, which can be applied toGram-negative bacteria and express industrial enzymes, such as lipaseand amydase, onto a cell surface in a fused form. In detail, the cellouter membrane protein complex participating in the synthesis ofpoly-γ-glutamate is composed of pgsA proteins, then the C-terminus ofthe pgsA protein gene is ligated with the N-terminus of a lipase oramydase among enzymes for industrial use.

EXAMPLES

Practical and presently preferred embodiments of the current inventionare illustrated in the following examples.

However, it is appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Construction of Surface Expression Vector pGNBCA

To prepare the surface expression vector of the current invention, whichuses the cell outer membrane protein gene pgsBCA participating in thesynthesis of poly-gamma-glutamate derived from a Bacillus sp. strain anda Gram-negative bacterium as the host cell, the total chromosomal DNA ofBacillus subtilis var. chungkookjang (accession number: KCTC 0697 BP)was purified.

The gene pgsBCA is composed of pgaB as a DNA fragment containing thenucleotide sequence of SEQ ID NO: 1, pgac as a DNA fragment containingthe nucleotide sequence of SEQ ID NO: 2, and pgaA as a DNA fragmentcontaining the nucleotide sequence of SEQ ID NO: 3 and has the aboveconsecutive nucleotide sequences.

To obtain the genes encoding the N-terminus and C-terminus of the cellinner membrane participating in the biosynthesis of poly-γ-glutamate,the total chromosome was adopted as the template and oligonucleotideswith the nucleotide sequence of SEQ ID NO: 4 (5-gaa cca tgg gct ggt tactcc tta tag cct g-3) at the N-terminus and nucleotide sequence of SEQ IDNO: 5 (5-ctc gga tcc ttt aga ttt tag ttt gtc act-3) at the C-terminusused as the primers. Then, a polymerase chain reaction (PCR) wasperformed using the template and primers.

The oligonucleotide primer of SEQ ID NO: 4 corresponding to theN-terminus was also constructed to contain the restriction enzyme NcoIrecognition site present in the expression vector pHCE19T(II), while theoligonucleotide primer of SEQ ID NO: 5 corresponding to the C-terminuswas constructed to contain the restriction enzyme BamHI recognition sitepresent in the expression vector pHCE19T(II). At this point, theamplified gene fragment was measured as having a size of 2.8 kb rangingfrom the N-terminal region of the cell outer membrane protein gene pgsBto the C-terminal region of pgsA. The gene fragment amplified throughthe PCR was digested with the restriction enzymes NcoI and BamHI andinserted into the constitutively high expression vector pHCE19T(II)digested with NcoI and BamHI. As a result, the new expression vectorcontaining the cell inner membrane protein gene that participates in thesynthesis of poly-γ-glutamate did not include a translation terminationcodon, contained a new additional restriction enzyme recognition site,was about 6.5 kb in size, had the nucleotide sequence of SEQ ID NO: 6,and was named expression vector pGNBCA (See FIG. 1).

The surface expression vector was transformed into Escherichia coli andthe resulting cell transformant deposited with the International DepositOrganization and Korean Collection for Type Cultures (KCTC: 52Eoeun-dong, Yusong-gu, Daejon) at the Korean Research Institute ofBioscience and Biotechnology (KRIBB) on Jul. 26, 2001 (accession number:KCTC 10025 BP).

Example 2 Construction of Surface Expression Vector pGNBCA-HB168

The recombinant expression vector pGNBCA-HB168, which can express anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen was constructed using the cell outermembrane protein gene pgsBCA participating in the synthesis ofpoly-γ-glutamate derived from a Bacillus sp. strain and a Gram-negativebacterium as the host cell.

To insert the hepatitis B virus S antigen gene into the surfaceexpression vector pGNBCA using a Gram-negative bacterium as the hostcell, the hepatitis B virus gene, about 1.4 kb in size, contained in thegeneral cloning vector pUC8 was adopted as the template andoligonucleotides with the nucleotide sequence of SEQ ID NO: 7 andnucleotide sequence of SEQ ID NO: 8 utilized as the primers. Then, apolymerase chain reaction (PCR) was performed using the template andprimers so as to amplify the S antigen gene. As a result, an amplifiedgene fragment 168 bp in size was obtained.

At this point, the primers with the nucleotide sequence of SEQ ID NO: 7and nucleotide sequence of SEQ ID NO: 8 were also constructed to containthe restriction enzyme BamHI and HindIII recognition sites. Then, theamplified S antigen gene of the hepatitis B virus was digested with therestriction enzymes BamHI and HindIII and ligated to the alreadyprepared C-terminal region of the cell outer membrane protein geneparticipating in the synthesis of poly-γ-glutamate by adjusting thetranslation codons. The resulting recombinant expression vectorpGNBCA-HB168 is illustrated in FIG. 1.

Example 3 Surface Expression of Antigenic Determinant FormingNeutralizing Antibodies Against Hepatitis B Virus S Antigen UsingRecombinant Expression Vector pGNBCA-HB168

The surface expression of an antigenic determinant forming neutralizingantibodies against the hepatitis B virus S antigen was examined usingthe recombinant expression vector pGNBCA-HB168.

The expression vector constructed in Example 2 was transformed into E.coli and cultivated in a 500 ml flask containing 50 ml of an LB medium(yeast extract 5 g/L, trypton 10 g/L, sodium chloride 5 g/L, pH 7.0) and100 mg/L of antibiotic ampicillin to induce surface expression.

The bacterial expression of the antigenic determinant formingneutralizing antibodies against the S antigen fused with the C-terminalgene participating in the synthesis of poly-γ-glutamate was identifiedby performing SDS-polyacrylamide gel electrophoresis and Westernimmunoblotting with antibodies against the S antigen. Essentially, theproteins obtained at the same cell concentration were denatured so thatexperimental samples could be prepared and then analyzed throughSDS-polyacrylamide gel electrophoresis so that the fractionated proteinswere transferred onto a PVDF membrane. The resulting PVDF membrane wasthen stirred in a blocking buffer (10 ml Tris HCl, 5% skim milk, pH 8.0)for one hour, blocked, and then reacted for 12 hours with a goat-derivedpolyclonal primary antibody against the S antigen diluted 1,000 timeswith the blocking buffer. After completing the reaction, the resultingmembrane was washed with the same buffer solution and reacted for 4hours with a secondary antibody conjugated with biotins and diluted1,000 times with the blocking buffer. The reacted membrane was thenwashed again with the buffer and immersed in an avidin-biotin reagentfor one hour and washed. The submersed membrane was colored by addingsubstrates and H202 and DAB reagents as dyes, which identified aspecific binding with the antibodies against the S antigen and abovefusion proteins (See FIG. 2, A). In FIG. 2, lane 1 is the untransformedhost cell JM109, while lane 2 is the cell transformantpGNBCA-HB168/JM109. As illustrated, a fusion protein band of about 48kDa was produced from the expression vector pGNBCA-HB168.

In addition, to directly confirm the expression of an antigenicdeterminant forming neutralizing antibodies against the hepatitis Bvirus S antigen situated on the E. coli surface, the E. Colitransformant inducing the surface expression was sonicated and separatedinto the soluble fraction, cell inner membrane fraction, and cell outermembrane fraction based on outer membrane fractionation, then analyzedby accomplishing SDS-polyacrylamide gel electrophoresis and Westernimmunoblotting of the antibodies against the S antigen. Essentially, theE. coli transformant used to induce the expression of the fusion proteinon the cell surface, as described above, and untransformed E. coli wereharvested, adjusted to the same concentration, and washed several timesusing a buffer solution (10 mM HEPES, pH 7.4). Thereafter, the resultantwas floated on a buffer containing 10 g/ml lysozyme, 1 mM PMSF, and 1 mMEDTA, reacted at 4° C. for 10 minutes, DNase (0.5 mg/ml) and RNase (0.5mg/ml) added, the mixture broken with a sonicator, and the intact E.coli and cellular debris separated at 4° C. for 20 minutes using10,000×g of centrifugation. The separated cellular debris of E. coli wasthen centrifuged at 4° C. for 20 minutes at 15,000×g and the fractionscontaining proteins of periplasm and cytoplasm collected. The resultingcell pellet was immersed in a PBS buffer (pH 7.4) containing 1% Sarcosyl(N-lauryl sarcosinate, sodium salt) and centrifuged at 4° C. for 2 hoursat 15,000×g and separated.

At this point, the supernatant was fractionated into the E. coli innermembrane and cell pellet of the E. coli outer membrane protein, thenanalyzed based on performing SDS-polyacrylamide gel electrophoresis andWestern blotting using antibodies against the S antigen. Among the aboveE. coli fractions, an antigenic determinant forming a neutralizingantibody against the S antigen was identified on the cell outer membrane(See FIG. 2; A: the result of E. coli membrane fraction after Westernblotting). As illustrated in FIG. 2, lane 1 is the untransformed E. coliJM 109 strain, lane 2 is the whole cell of the E. coli transformantpGNBCA-HB168/JM109, lane 3 is the soluble fraction of the E. colitransformant pGNBCA-HB168/JM109, lane 4 is the cell inner membranefraction of the E. coli transformant pGNBCA-HB168/JM109, and lane 5 isthe cell outer membrane fraction of the E. coli transformantpGNBCA-HB168/JM109.

An antigenic determinant forming a neutralizing antibody against Santigen was verified as having been expressed onto the E. coli cellsurface from the C-terminus of the poly-γ-glutamate synthetase proteinby performing fluorescence activating cell sorting (FACS) flowcytometry. For immunofluorescence staining, the E. coli used to inducethe surface expression was harvested at the same cell concentration andwashed several times using a PBS buffer (pH 7.4). The resulting cellpellet was then suspended using 1 ml of a buffer containing 1% bovineserum albumin and reacted with goat-derived polyclonal primaryantibodies against the S antigen diluted 1,000 times at 4C for 12 hours.After completing the reaction, the resulting cells were washed againseveral times, suspended using 1 ml of a buffer containing 1% bovineserum albumin, then reacted at 4C for 3 hours with biotin-associatedsecondary antibodies against the S antigen diluted to 1,000 times. Also,the completely reacted cells were washed several times using a buffersolution, suspended with 0.1 ml of a buffer containing 1% bovine serumalbumin, then bound with the streptoavidin-R-phycoerythrin dyeingreagent diluted to 1,000 times, which is specific for biotins.

Thereafter, the E. coli cells were rewashed several times and assayed byperforming fluorescence activating cell sorting flow cytometry. As aresult, an antigenic determinant protein forming a neutralizing antibodyagainst the S antigen was confirmed as having been expressed onto thecell surface, as distinct from the untransformed E. coli (See FIG. 2,B). As illustrated in FIG. 2, the white band depicts the untransformedE. coli JM109 strain, while the black band is derived from the E. colitransformant pGNBCA-HB168/JM109. Consequently, no antigenic determinantprotein forming a neutralizing antibody against the S antigen wasexpressed from the untransformed E. coli strain, yet obviouslydetermined from the E. coli transformant transformed with the surfaceexpression vector of the current invention.

Example 4 Construction of Recombinant Surface Expression VectorpGNCA-HB101 and Surface Expression of Antigenic Determinant FormingNeutralizing Antibody Against Hepatitis B Virus S Antigen

(1) A recombinant expression vector that can express an antigenicdeterminant forming a neutralizing antibody against the hepatitis Bvirus S antigen was constructed using the cell outer membrane proteingene pgsBCA participating in the synthesis of poly-γ-glutamate derivedfrom a Bacillus sp. strain and a Gram-negative bacterium as the hostcell.

The gene pgsCA has a consecutive nucleotide sequence containing the pgsCDNA of SEQ ID NO: 2 and pgaA DNA of SEQ ID NO: 3.

To obtain the N-terminal and C-terminal genes encoding the pgsc and pgsAproteins from the cell outer membrane protein gene participating in thesynthesis of poly-γ-glutamate, the total chromosome was utilized as thetemplate and oligonucleotides containing the nucleotide sequences of SEQID NO: 9 at the N-terminus and SEQ ID NO: 5 at the C-terminus used asthe primers for performing a polymerase chain reaction.

The primer corresponding to the N-terminus and containing the sequenceof SEQ ID. NO: 9 was also constructed to include the restriction enzymeNdeI recognition site. At this point, the amplified gene region includedabout 1.6 kb from the N-terminal region of the outer membrane proteingene pgsC participating in the synthesis of poly-γ-glutamate to theC-terminal region of pgsA.

The genes amplified through the polymerase chain reaction were digestedwith the restriction enzymes NdeI and BamHI and inserted into theconstitutively high expression vector pHCE19T(II) already digested withBamHI and NdeI, thereby creating a new expression vector, about 5.3 kbin size, with new restriction enzyme recognition sites, and notermination codon at the end of the cell outer membrane protein gene,called expression vector pGNCA (See FIG. 3).

(2) The recombinant expression vector pGNCA-HB168 that can express anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen was constructed through the same procedureas described above in Example 2. In detail, the recombinant expressionvector was prepared by exploiting the cell outer membrane protein genespgsc and pgsA from the pgsBCA gene participating in the synthesis ofpoly-γ-glutamate and a Gram-negative bacterium as the host cell.

The recombinant expression vector pGNCA-HB168 constructed above isdepicted in FIG. 3.

(3) The expression of an antigenic determinant forming a neutralizingantibody against the hepatitis B virus S antigen using the surfaceexpression vector pGNCA-HB168 was examined as follows:

The surface expression vector was transformed into a E. coli host celland expression induced using the same procedure as described above inExample 3. Then, an antigenic determinant forming a neutralizingantibody against the S antigen fused with the cell outer membraneprotein pgsCA was identified as having been expressed in the E. colitransformant based on performing SDS-polyacrylamide gel electrophoresisand Western blotting using antibodies against the S antigen (See FIG.4).

As illustrated in FIG. 4, lane 1 is the untransformed host cell JM109,while lane 2 is the cell transformant pGNCA-HB168/JM109. As a result,the fused protein expressed from the recombinant expression vectorpGNCA-HB168 was identified as a band of about 48 kDa.

Example 5 Construction of Recombinant Surface Expression VectorpGNA-HB168 and Surface Expression of Antigenic Determinant FormingNeutralizing Antibody Against Hepatitis B Virus S Antigen

(1) A recombinant expression vector that can express an antigenicdeterminant forming a neutralizing antibody against the hepatitis Bvirus S antigen was constructed using the cell outer membrane proteingene pgsA from the pgsBCA gene participating in the synthesis ofpoly-γ-glutamate derived from a Bacillus sp. strain and a Gram-negativebacterium as the host cell.

The gene pgsA contains the nucleotide sequence of SEQ ID NO: 3.

To obtain the N-terminal and C-terminal genes encoding the pgsA proteinfrom the cell outer membrane protein gene participating in the synthesisof poly-γ-glutamate, the total chromosome was utilized as the templateand oligonucleotides containing the nucleotide sequences of SEQ ID NO:10 at the N-terminus and SEQ ID NO: 5 at the C-terminus used as theprimers for performing a polymerase chain reaction.

The primer corresponding to the N-terminus and containing the sequenceof SEQ ID. NO: 10 was also constructed to include the restriction enzymeNdeI recognition site. At this point, the amplified gene region wasabout 1.1 kb from the N-terminal region of the outer membrane proteingene pgsA participating in the synthesis of poly-γ-glutamate to theC-terminal region of pgsA.

The genes amplified through the polymerase chain reaction were digestedwith the restriction enzymes NdeI and BamHI and inserted into theconstitutively high expression vector pHCE19T(II) already digested withBamHI and NdeI, thereby creating a new expression vector, about 4.8 kbin size, with new restriction enzyme recognition sites, and notermination codon at the end of the cell outer membrane protein gene,called expression vector pGNCA (See FIG. 5).

(2) The recombinant expression vector pGNCA-HB168 that can express anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen was constructed through the same proceduredescribed above in Example 2. In detail, the recombinant expressionvector was prepared by exploiting the cell outer membrane protein genepgsA from the pgsBCA gene participating in the synthesis ofpoly-γ-glutamate and a Gram-negative bacterium as the host cell.

The recombinant expression vector pGNA-HB168 constructed above isdepicted in FIG. 5.

(3) The expression of an antigenic determinant forming a neutralizingantibody against the hepatitis B virus S antigen using the surfaceexpression vector pGNA-HB168 was examined as follows:

The surface expression vector was transformed into the E. coli host celland expression induced through the same procedure as described above inExample 3. Then, an antigenic determinant forming a neutralizingantibody against the S antigen fused with the cell outer membraneprotein pgsCA was identified as having been expressed in the E. colitransformant by performing SDS-polyacrylamide gel electrophoresis andWestern blotting using antibodies against the S antigen (See FIG. 5).

As illustrated in FIG. 5, lane 1 is the untransformed host cell JM109,while lane 2 is the cell transformant pGNA-HB168/JM109. As a result, thefused protein expressed from the recombinant expression vectorpGNA-HB168 was identified as a band of about 48 kDa.

Furthermore, the expression of an antigenic determinant forming aneutralizing antibody against S antigen onto the E. coli cell surfacefrom the cell outer membrane protein pgsA was also verified based onperforming fluorescence activating cell sorting (FACS) flow cytometry.For this purpose, the same procedure as described above in Example 3 wasused, and an antigenic determinant forming a neutralizing antibodyagainst the S antigen was detected on the cell surface, as distinct fromthe results for the untransformed E. coli(See FIG. 4). As illustrated inFIG. 4, the white band is the untransformed E. coli JM109, while theblack band is derived from the E. coli transformant pGNA-HB168/JM109. Asa result, the untransformed E. coli did not exhibit the expression of anantigenic determinant forming a neutralizing antibody against the Santigen, whereas the E. coli transformant transformed with the surfaceexpression vector containing only the pgsA gene clearly revealed theexpression of an antigenic determinant forming a neutralizing antibodyagainst the S antigen onto the cell surface.

Example 6 Construction of Recombinant Surface Expression Vector pGNHB-Aand Surface Expression of Antigenic Determinant Forming NeutralizingAntibody Against Hepatitis B Virus S Antigen

(1) A recombinant expression vector that can express an antigenicdeterminant forming a neutralizing antibody against the hepatitis Bvirus S antigen was constructed using the N-terminal pgsA gene from thecell outer membrane protein gene pgsBCA participating in the synthesisof poly-γ-glutamate derived from a Bacillus sp. strain and aGram-negative bacterium as the host cell.

The gene pgsA contains the nucleotide sequence of SEQ ID NO: 3.

To obtain the N-terminal and C-terminal genes encoding the pgsA proteinfrom the cell outer membrane protein gene participating in the synthesisof poly-γ-glutamate, the total chromosome was utilized as the templateand oligonucleotides containing the nucleotide sequences of SEQ ID NO:11 at the N-terminus and SEQ ID NO: 12 at the C-terminus used as theprimers for performing a polymerase chain reaction.

The primer corresponding to the N-terminus and containing the sequenceof SEQ ID. NO: 11 was also constructed to include the restriction enzymeBamHI recognition site. At this point, the amplified gene region wasabout 1.1 kb from the N-terminal region of the outer membrane proteingene pgsA participating in the synthesis of poly-γ-glutamate to theC-terminal region of pgsA. The genes amplified through the polymerasechain reaction were digested with the restriction enzymes BamHI andHindIII and inserted into the constitutively high expression vectorpHCE19T(II) already digested with BamHI and HindIII, thereby creating anew expression vector called pGNA2, about 4.8 kb in size, with newrestriction enzyme recognition sites at the front of the pgsA gene, andno termination codon at the end of the cell outer membrane protein geneparticipating in the synthesis of poly-γ-glutamate (See FIG. 6).

(2) The recombinant expression vector pGNHB-A that can express anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen was also constructed. In detail, therecombinant expression vector was prepared by exploiting the N-terminalsequence of the cell outer membrane protein gene pgsA from the proteingene pgsBCA participating in the synthesis of poly-γ-glutamate and aGram-negative bacterium as the host cell.

To insert the hepatitis B virus S antigen gene into the surfaceexpression vector pGNA2, while exploiting a Gram-negative bacterium asthe host cell, about 1.4 kb of the hepatitis virus gene cloned into thegeneral cloning vector pUC18 was adopted as the template andoligonucleotides with the nucleotide sequences of SEQ ID NO: 13 and SEQID NO: 14 utilized as the primers for amplifying the S antigen genethrough a polymerase chain reaction. At this moment point, the amplifiedgene region was 1.6 bp in size.

The primers with nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14were also constructed to contain the restriction enzyme NdeI and BamHIrecognition sites present in the surface expression vector pGNA2. Theamplified genes of the hepatitis B virus S antigen were digested withthe restriction enzymes NdeI and BamHI and ligated to the N-terminalregion of the pgsA gene from among the cell outer membrane genesparticipating in the synthesis of poly-γ-glutamate in the surfaceexpression vector pGNA2 already prepared by adjusting the translationcodons. The recombinant expression vector pGNHB-A constructed above isdepicted in FIG. 6.

(3) The expression of an antigenic determinant forming a neutralizingantibody against the hepatitis B virus S antigen using the surfaceexpression vector pGNHB-A was examined as follows:

The surface expression vector was transformed into the E. coli host celland expression induced using the same procedure described above inExample 3. Then, the identification of an antigenic determinant forminga neutralizing antibody against the S antigen fused with the cell outermembrane protein pgsCA expressed in the E. coli transformant wasperformed based on SDS-polyacrylamide gel electrophoresis and Westernblotting using antibodies against the S antigen (See FIG. 5).

As illustrated in FIG. 5, lane 1 is the untransformed host cell JM109,while lane 2 is the cell transformant pGNHB-A/JM109. As a result, thefused protein expressed from the recombinant expression vector pGNHB-Awas identified as a band of about 48 kDa.

Example 7 Construction of Recombinant Surface Expression VectorpGNC-PreS1 and Surface Expression of Antigenic Determinant FormingNeutralizing Antibody Against Hepatitis B Virus PreS1 Antigen

(1) A recombinant expression vector that can express an antigenicdeterminant forming a neutralizing antibody against the hepatitis Bvirus S antigen was constructed using the C-terminal pgsc gene from thecell outer membrane protein gene pgsBCA participating in the synthesisof poly-γ-glutamate derived from a Bacillus sp. strain and aGram-negative bacterium as the host cell.

The gene pgsC contains the nucleotide sequence of SEQ ID NO: 2.

To obtain the N-terminal and C-terminal genes encoding the pgsA proteinfrom among cell outer membrane proteins participating in the synthesisof poly-γ-glutamate, the total chromosome was utilized as the templateand oligonucleotides containing the nucleotide sequences of SEQ ID NO:15 at the N-terminus and SEQ ID NO: 16 at the C-terminus used as theprimers for performing a polymerase chain reaction.

The primer corresponding to the N-terminus and containing the sequenceof SEQ ID. NO: 15 was also constructed to include the restriction enzymeNdeI recognition site present in the surface expression vector pHCE19T(II), while the primer containing the sequence of SEQ ID. NO: 16 wasconstructed to include the restriction enzyme BamHI recognition sitepresent in the surface expression vector pHCE 19T(II). At this point,the amplified gene region was about 0.45 kb in size from the N-terminalregion of the outer membrane protein gene pgsC participating in thesynthesis of poly-γ-glutamate to the C-terminal region of pgsC. Thegenes amplified through the polymerase chain reaction were digested withthe restriction enzymes BamHI and NdeI and inserted into theconstitutively high expression vector pHCE19T(II) already digested withBamHI and NdeI, thereby creating a new expression vector called pGNC,about 4.1 kb in size, with new restriction enzyme recognition sites, andno termination codon at the end of the cell outer membrane protein geneparticipating in the synthesis of poly-γ-glutamate (See FIG. 7).

(2) The recombinant expression vector pGNC-PreS1 that can express anantigenic determinant forming a neutralizing antibody against thehepatitis B virus PreS1 surface antigen was also constructed. In detail,the recombinant expression vector was prepared by exploiting theN-terminal sequence of the cell outer membrane protein gene pgsc fromthe protein gene pgsBCA participating in the synthesis ofpoly-γ-glutamate and using a Gram-negative bacterium as the host cell.

To insert the hepatitis B virus PreS1 antigen gene into the surfaceexpression vector pGNC, while exploiting a Gram-negative bacterium asthe host cell, about 1.5 kb of the hepatitis virus gene cloned into thegeneral cloning vector pUC18 was adopted as the template andoligonucleotides with the nucleotide sequences of SEQ ID NO: 17 and SEQID NO: 18 utilized as the primers for amplifying the S antigen genethrough a polymerase chain reaction. At this point, the amplified generegion became 356 bp in size.

The primers with the nucleotide sequences of SEQ ID NO: 17 and SEQ IDNO: 18 were also constructed to contain the restriction enzyme HindIIIand BamHI recognition sites present in the surface expression vectorpGNC. The amplified genes of the hepatitis B virus PreS1 antigen weredigested with the restriction enzymes HindIII and BamHI and ligated tothe N-terminal region of the pgsc gene from the cell outer membrane geneparticipating in the synthesis of poly-γ-glutamate in the surfaceexpression vector pGNC already prepared by adjusting the translationcodons. The recombinant expression vector pGNC-PreS1 constructed aboveis depicted in FIG. 7.

(3) The expression of an antigenic determinant forming a neutralizingantibody against the hepatitis B virus S antigen using the surfaceexpression vector pGNC-PreS1 was examined as follows:

The surface expression vector was transformed into the E. coli host celland expression induced using the same procedure described above inExample 3. Then, the identification of an antigenic determinant forminga neutralizing antibody against the PreS1 antigen fused with the cellouter membrane protein pgsc expressed in the E. coli transformant wasperformed based on SDS-polyacrylamide gel electrophoresis and Westernblotting using antibodies against the PreS1 antigen (See FIG. 8).

As illustrated in FIG. 8, lane 1 is the untransformed host cell JM109,while lanes 2 and 3 cell and the transformant pGNC-PreS1/JM109. As aresult, the fused protein expressed from the recombinant expressionvector pGNC-PreS1 was identified as a band of about 27 kDa.

Example 8 Construction of Recombinant Surface Expression VectorspHCE1LB:BCA and pHCE1LB:BCA-HB168, and Surface Expression of AntigenicDeterminant Forming Neutralizing Antibody Against Hepatitis B Virus SAntigen

(1) The recombinant expression vector pHCE1LB:BCA that can express anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen onto a cell surface was constructed usingthe C-terminal pgsC gene from the cell outer membrane protein genepgsBCA participating in the synthesis of poly-γ-glutamate derived from aBacillus sp. strain and a Gram-negative bacterium as the host cell.

The plasmid pHCE1LB was adopted as the cloning vector as it canreplicate and be selected in both Gram-negative and Gram-positivebacteria. The expression vector pHCE1LB was comprised of aconstitutively high expressing HCE promoter, plus the cloning sitecontained various restriction enzyme recognition sites, originsreplicable in Gram-negative bacteria, and antibiotic-resistant markersof ampicillin. In addition, the expression vector pHCE1LB was composedof origins replicable in Gram-positive bacteria derived fromLactobacillus sp. and antibiotic-resistant markers of chloroamphenicol.

To obtain the N-terminal and C-terminal genes encoding the pgsBCAprotein from among the cell outer membrane proteins participating in thesynthesis of poly-γ-glutamate, the total chromosome was utilized as thetemplate and oligonucleotides containing the nucleotide sequences of SEQID NO: 4 at the N-terminus and SEQ ID NO: 5 at the C-terminus used asthe primers for performing a polymerase chain reaction. At this point,the amplified gene region was about 2.8 kb in size from the N-terminalregion of the outer membrane protein gene pgsB participating in thesynthesis of poly-gamma-glutamate to the C-terminal region of pgsA. Thegenes amplified through the polymerase chain reaction were digested withthe restriction enzymes NcoI and BamHI and inserted into the expressionvector pHCE1LB already digested with BamHI and NcoI, thereby creating anew expression vector called pHCE1LB:BCA, about 8 kb in size, with newrestriction enzyme recognition sites, no termination codon at the end ofthe cell outer membrane protein gene participating in the synthesis ofpoly-γ-glutamate, and utilizing both Gram-negative and Gram-positivebacteria as a host cell (See FIG. 9).

(2) The recombinant expression vector pHCE1LB:BCA-HB168 that can expressan antigenic determinant forming a neutralizing antibody against thehepatitis B virus S surface antigen was also constructed. In detail, therecombinant expression vector was prepared using the same proceduredescribed above by exploiting the cell outer membrane protein genepgsBCA participating in the synthesis of poly-γ-glutamate and using aGram-negative bacterium as the host cell.

The recombinant expression vector pHCE1LB:BCA-HB168 constructed above isillustrated in FIG. 9.

(3) The expression of an antigenic determinant forming a neutralizingantibody against the hepatitis B virus S antigen using the surfaceexpression vector pHCE1LB:BCA-HB168 and Salmonella typhi Ty21a as theGram negative host was investigated as follows:

The surface expression vector was transformed into the Salmonella typhiTy21a host cell and expression induced using the same proceduredescribed above in Example 3. Then, the identification of an antigenicdeterminant expressed onto the cell surface of Salmonella typhi Ty21awas conducted based on the outer membrane fractionation method byseparating the soluble fraction, inner membrane fraction, and outermembrane fraction and performing SDS-polyacrylamide gel electrophoresisand Western immunoblotting using antibodies against the S antigen. As aresult, the production of an antigenic determinant forming aneutralizing antibody against the S antigen fused with pgsA,corresponding to a 48 kDa band, was confirmed to be situated in theouter membrane fraction among the Salmonella typhi Ty21a fractions (SeeFIG. 10). As illustrated in FIG. 10, lane 1 is the untransformed hostcell, lane 2 is the whole cell of the cell transformant, and lanes 3, 4,and 5 are the soluble fraction, inner membrane fraction, and outermembrane fraction, respectively, of the cell transformant.

In addition, the surface expression of an antigenic determinant forminga neutralizing antibody against the S antigen was verified by performingfluorescence activating cell sorting (FACS) flow cytometry. For thispurpose, the same procedure was used as described above in Example 3,which revealed an antigenic determinant protein on the cell surface, asdistinct from the result for the untransformed Salmonella typhi Ty21a(See FIG. 10). As illustrated in FIG. 10, black without an arrow is theuntransformed Salmonella typhi Ty21a, while black with an arrow is thetransformed Salmonella typhi Ty21a. As a result, the E. colitransformant transformed with the surface expression vector clearlyexhibited the expression of an antigenic determinant forming aneutralizing antibody against the S antigen onto the cell surface.

Example 9 Analysis of Vaccine Efficacy in Mirobes Expressing AntigenicDeterminant Forming Neutralizing Antibody Against Hepatitis B Virus SAntigen onto Cell Surface

The recombinant vector pHCE1LB:BCA-HB168 constructed for surfaceexpression in Example 8 was transformed into the Gram-negativebacterium, Salmonella typhi Ty21a, and expression induced onto the cellsurface using the same procedure as described in Example 3. Thereafter,the antigenicity of the antigenic determinant forming a neutralizingantibody against the hepatitis B virus S antigen fused with the cellouter membrane protein participating in the synthesis ofpoly-γ-glutamate was measured.

Essentially, the recombinant vector pHCE1LB:BCA-HB168 for surfaceexpression was transformed into Salmonella typhi Ty21a and theexpression of the above antigens examined. Thereafter, some of theSamonella strain was administered to the nasal cavity of BALB/c mice,then after several days, (1) the blood serum of the mice was collectedand the presence of IgG antibodies against the S antigen examined in theserum, and (2) the organs of the mice were collected, then the presenceof IgA antibodies against the S antigen in the suspension solution usedto wash the organs was investigated using an enzyme-linked immunosorbentassay (ELISA).

At this point, the harvested cells were washed several times using a PBSbuffer (pH 7.4) and adjusted to the same cell concentration, then 2×10⁹of Salmonella typhi Ty21a on which the antigens had beensurface-expressed was administered to the nasal cavity of 4˜6 week-oldBALB/c mice twice with a 3-day interval in between. After 4 weeks, twofurther injections were administered with a 3-day interval in between,then, 2 weeks later the blood serum of the mice and solution used towash the organs were collected and measured for their antibody titersagainst the antigens based on the ELISA method using the S antigen (SeeFIG. 11).

As illustrated in FIG. 11, graph A shows the IgG antibody titers againstthe S antigenic determinant in the blood serum: i.e. the titers of theuntransformed Salmonella, titers of the Samonella transformed with theexpression vector pHCE1LB:BCA-HB168, and titers of the Salmonellatransformed with the expression vector pHCE1LB:HB168. In FIG. 11, graphB shows the IgA antibody titers against the S antigenic determinant inthe organs: i.e. the titers of untransformed Salmonella, titers of theSalmonella transformed with the expression vector pHCE1LB:HB168, andtiters of the Salmonella transformed with the expression vectorpHCE1LB:BCA-HB168.

As demonstrated in FIG. 11, the blood serum and solution used to washthe organs from the BALB/c mice group administered with the Salmonellatyphi Ty21a transformant transformed with the surface expression vectorpHCE1LB:BCA-HB168 exhibited much higher antibody titers for IgG and IgA,as distinct from the results of the other groups.

Accordingly, the microbes of the current invention expressing anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen onto a cell surface were confirmed to workas an effective live vaccine.

Example 10 Surface Expression of Antigenic Determinant FormingNeutralizing Antibody Against Hepatitis B Virus S Antigen onto GramPositive Bacterium Transformed with Expression Vector pHCE1LB:BCA-HB168

The surface expression of an antigenic determinant forming aneutralizing antibody against the hepatitis B virus S antigen onto theGram-positive bacterium Lactobacillus casei transformed with therecombinant vector pHCE1LB:BCA-HB168 constructed for surface expression,as described in Example 8, was investigated.

The recombinant vector used for the surface expression, as preparedabove, was transformed into Lactobacillus casei and cultivated in astatic state in a 500 ml flask containing 200 ml of an MRS medium, plus50 mg/L of the antibiotic chloroamphenicol to induce the surfaceexpression.

To locate the antigenic determinant situated on the cell surface ofLactobacillus casei, the cell transformants were separated using thecell wall fractionation method into fractions, such as the cellcytoplasm and cell wall etc., and analyzed by performingSDS-polyacrylamide gel electrophoresis and Western blotting usingantibodies against the S antigen. Essentially, the Lactobacillustransformant induced to express the fused proteins onto the cell surfaceand untransformed Lactobacillus were harvested based on the same cellconcentration, washed several times using a TES buffer (10 mM Tris-HCl,pH 8.0, 1 MM EDTA, 25% sucrose), suspended using distilled watercontaining 5 mg/ml lysozyme, 1 mM PMSF, and 1 mM EDTA, then frozen anddissolved at −60° C. and room temperature, respectively, several times.The resulting cells were disrupted using a sonicator by adding 0.5 mg/mlof DNase and 0.5 mg/ml of RNase. Thereafter, the sonicated cell solutionwas centrifuged at 4C for 20 minutes at 10,000×g for separation into awhole Lactobacillus pellet (whole cell fraction), i.e. not sonicated,and cellular debris (supernatant), then centrifuged again at 4C for onehour at 21,000×g to collect a supernatant containing the cytoplasmicproteins of Lactobacillus and a cell pellet. The resulting cell pelletwas suspended in a TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 7.4)containing 1% SDS to collect the cell wall proteins (cell wallfractions) of Lactobacillus.

Each fraction was analyzed to confirm the sites on the cell wall wherean antigenic determinant formed a neutralizing antibody against the Santigen by performing SDS-polyacrylamide gel electrophoresis and Westernimmunoblotting using antibodies against the S antigen (See FIG. 12A). Asillustrated in FIG. 12, lanes 1, 3, and 5 are the untransformedLactobacillus casei, lane 2 is the whole cell of Lactobacillus caseitransformed with the expression vector pHCE1B:BCA-HB168, and lanes 4 and6 are the soluble fraction and cell wall fraction, respectively, of thetransformed cell.

In addition, the surface expression of the antigenic determinant forminga neutralizing antibody against the S antigen from the C-terminus of thepoly-gamma-glutamate synthetase protein was verified by performingfluorescence activating cell sorting (FACS) flow cytometry. For thispurpose, the same procedure was used as described for Example 3, whichrevealed an antigenic determinant protein on the cell surface, asdistinct from the result for the untransformed Lactobacillus (See FIG.12B). As illustrated in FIG. 12B, the white band is the untransformedLactobacillus, while the black band is derived from the Lactobacillustransformed with the expression vector pHCE1LB:BCA-HB168. As a result,the Lactobacillus transformant transformed with the surface expressionvector clearly exhibited the expression of an antigenic determinantforming a neutralizing antibody against the S antigen onto the cellsurface.

Example 11 Analysis of Vaccine Efficacy 2 in Microbes ExpressingAntigenic Determinant Forming Neutralizing Antibody Against Hepatitis BVirus S Antigen onto Cell Surface

The recombinant vector pHCE1LB:BCA-HB168 constructed for surfaceexpression in Example 8 was transformed into the Gram-positive bacteriumLactobacillus casei and expression induced onto the cell surface usingthe same procedure as described in Example 3. Thereafter, theantigenicity of the antigenic determinant forming a neutralizingantibody against the hepatitis B virus S antigen fused with the cellouter membrane protein participating in the synthesis ofpoly-γ-glutamate was measured.

Essentially, the recombinant vector pHCE1LB:BCA-HB168 created forsurface expression was transformed into Lactobacillus casei, harvestedto reach the same cell concentration, and washed several times using aPBS buffer (pH 7.4). Then, 5×10¹⁰ cells of Lactobacillus on which theantigens had been surface-expressed were administered to the oral cavityof BALB/c mice 4˜6 weeks of age three times a day interval in between,then after 4 weeks 3 more injections were given one day apart. Inaddition, 1×10¹⁰ cells of Lactobacillus on which the antigens had beensurface-expressed were administered to the nasal cavity of BALB/c micetwice with a 3-day interval in between and after 4 weeks 2 furtherinjections with a 3-day interval in between. Two weeks after the oraland nasal administrations, the (1) blood serum of the mice was collectedand the IgG antibody titers against the S antigen examined, and (2) theorgans of the mice were collected and the IgA antibody titers againstthe S antigen investigated in the suspension solution used to wash thethe organ using an enzyme-linked immunosorbent assay (ELISA)(See FIG.13).

As illustrated in FIG. 13, graph A shows the IgG antibody titers againstthe S antigenic determinant for the blood serum: i.e. the titers of theLactobacillus transformed with the expression vector pHCE1LB:BCA-HB168in the nasal administered group, titers of the Lactobacillus transformedwith the expression vector pHCE1LB:BCA-HB168 in the oral administeredgroup, titers of the Lactobacillus transformed with the expressionvector pHCE1LB:HB168 (in which the pgsBCA gene is deleted and from whichHB168 can be expressed in the cells) in the oral administered group, andtiters of the untransformed Lactobacillus in the oral administeredgroup. In FIG. 13, graph B shows the IgA antibody titers against the Santigenic determinant for the organs: i.e. the titers of theuntransformed Lactobacillus, titers of the Lactobacillus transformedwith the expression vector pHCE1LB:HB168, titers of the Lactobacillustransformed with the expression vector pHCE1LB:BCA-HB168. Theexperiments were performed using separate groups: nasal administeredgroup and oral administered group.

As demonstrated in FIG. 11, the blood serum and solution used to washthe organs from the BALB/c mice group administered the Lactobacillustransformant transformed with the surface expression vectorpHCE1LB:BCA-HB168 exhibited much higher antibody titers for IgG and IgA,as distinct from the results for the comparative groups.

Accordingly, the microbes of the current invention expressing anantigenic determinant forming a neutralizing antibody against thehepatitis B virus S antigen onto a cell surface were confirmed to workas an effective live vaccine.

Example 12 Construction of Surface Expression Vector pHCE1LB:A

A recombinant expression vector that can express an antigenicdeterminant forming a neutralizing antibody against the hepatitis Bvirus S antigen was constructed using only the cell outer membraneprotein gene pgsA from the pgsBCA gene participating in the synthesis ofpoly-γ-glutamate derived from a Bacillus sp. Strain: i.e. the surfaceexpression vector pHCE1LB:A, which can exploit Gram-negative orGram-positive bacteria as the host cell.

To obtain the N-terminal and C-terminal genes encoding the pgsA proteinamong the cell outer membrane proteins participating in the synthesis ofpoly-γ-glutamate, the total chromosome was utilized as the template andoligonucleotides containing the nucleotide sequences of SEQ ID NO: 10 atthe N-terminus and SEQ ID NO: 5 at the C-terminus used as the primersfor performing a polymerase chain reaction.

At this point, the amplified gene region was about 1.1 kb in size fromthe N-terminal region of the outer membrane protein gene pgsAparticipating in the synthesis of poly-γ-glutamate to the C-terminalregion. The genes amplified through the polymerase chain reaction weredigested with the restriction enzymes NdeI and BamHI and inserted intothe expression vector pHCE1LB already digested with BamHI and NdeI,thereby creating a new expression vector, called pGNCA, about 6.3 kb insize, with new restriction enzyme recognition sites, no terminationcodon at the end of the cell outer membrane protein gene, and theability to exploit both Gram-negative and Gram-positive bacteria as ahost cell (See FIG. 14).

Example 13 Construction of Recombinant Surface Expression VectorpHCE1LB:A-TGEN1 and Surface Expression of TGE N Antigen

The recombinant expression vector pHCE1LB:A-TGEN1 that can express anucleoprotein (N) antigen of the transmissible gastroenteritis virus(TGE) inducing porcine transmissible gastric diseases onto a cellsurface was constructed using the pgsA gene from the cell outer membraneprotein gene pgsBCA participating in the synthesis of poly-γ-glutamatederived from a Bacillus sp. strain and Gram-negative or Gram positivebacteria as the host cell.

To introduce the major antigenic determinant region of the N antigengenes of the TGE virus to the surface expression vector pHCE1LB:A, asprepared in Example 12, using Gram-negative or Gram-positive bacteria asthe host cell, about 1.1 kb of the TGE virus gene was cloned into thegeneral cloning vector pUC8 and adopted as the template andoligonucleotides with the nucleotide sequences of SEQ ID NO: 19 and SEQID NO: 20 utilized as the primer for performing a polymerase chainreaction (PCR) to amplify the S antigen gene. As a result, an amplifiedgene fragment 415 bp in size was obtained. At this point, the primerswith the nucleotide sequences of SEQ ID NO: 19 and SEQ ID NO: 20 werealso constructed to contain the restriction enzyme BamHI and HindIIIrecognition sites present in the surface expression vector pHCE1LB:A.

Thereafter, the amplified N antigen gene of the TGE virus was digestedwith the restriction enzymes BamHI and HindIII and ligated to thealready prepared C-terminal region of the cell outer membrane proteingene pgsA in the surface expression vector pHCE1LB:A by adjusting thetranslation codons. The resulting recombinant expression vectorpHCE1LB:A-TGEN1 is illustrated in FIG. 1.

The surface expression of the TGE virus N antigen was investigated withE. coli and Lactobacillus using the recombinant expression vectorpHCE1LB:A-TGEN1. For this purpose, the recombinant expression vector wastransformed into E. coli and Lactobacillus and expression inducedthrough the same procedure as described above. The expression of the TGEN antigen fused with the cell outer membrane protein onto the cellsurface was then confirmed by performing SDS-polyacrylamide gelelectrophoresis and Western immunoblotting using an antibody to the TGEN antigen and pgsA protein, respectively (See FIG. 15).

As illustrated in FIG. 15, in A, lane 1 is the untransformed host cellJM109 and lanes 2 and 3 are the cell transformant pHCE1LB:A-TGEN1/JM109,while in B, lane 1 is the untransformed host cell Lactobacillus casei,and lanes 3 and 4 are the Lactobacillus casei transformantpHCE1LB:A-TGEN1. As a result, the band corresponding to the fusedprotein produced from the expression vector pHCE1LB:A-TGEN1 wasidentified as about 57 kDa in size.

Example 14 Construction of Recombinant Surface Expression VectorpHCE1LB:A-PEDN and Surface Expression of PED N Antigen

(1) The recombinant expression vector pHCE1LB:A-PEDN that can express anucleoprotein (N) antigen of the porcine epidemic diarrhea virus (PED)inducing porcine transmissible gastric diseases onto a cell surface wasconstructed using the pgsA gene from the cell outer membrane proteingene pgsBCA participating in the synthesis of poly-γ-glutamate derivedfrom a Bacillus sp. strain and Gram-negative or Gram-positive bacteriaas the host cell.

To introduce the antigenic determinant region of N antigen genes of thePED virus into the surface expression vector pHCE1LB:A, as prepared inExample 12, using Gram-negative or Gram-positive bacteria as the hostcell, about 1.3kb of the PED virus gene was cloned into the generalcloning vector pUC8 and adopted as the template and oligonucleotideswith the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 22utilized as the primers for performing a polymerase chain reaction (PCR)to amplify the S antigen gene. As a result, an amplified gene fragment1326 bp in size was obtained. At this point, the primers with thenucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 22 were alsoconstructed to contain the restriction enzyme BamHI and HindIIIrecognition sites present in the surface expression vector pHCE1LB:A.

Thereafter, the amplified N antigen gene of the PED virus was digestedwith the restriction enzymes BamHI and HindIII and ligated to thealready prepared C-terminal region of the cell outer membrane proteingene pgsA in the surface expression vector pHCE1LB:A by adjusting thetranslation codons. The resulting recombinant expression vectorpHCE1LB:A-PEDN is illustrated in FIG. 14.

(2). The surface expression of the PED virus N antigen onto E. coli andLactobacillus was investigated using the recombinant expression vectorpHCE1LB:A-PEDN. For this purpose, the recombinant expression vector wastransformed into E. coli and Lactobacillus and expression inducedthrough the same procedure as described above. Then, the expression ofthe PED N antigen fused with the cell outer membrane protein pgsA ontothe cell surface was confirmed by performing SDS-polyacrylamide gelelectrophoresis and Western immunoblotting using an antibody against thePED N antigen and pgsA protein, respectively (See FIG. 16).

As illustrated in FIG. 16, in A, lane 1 is the untransformed host cellJM109, and lanes 2 and 3 are the cell transformantpHCE1LB:A-TGEN1/JM109, while in B, lane 1 is the untransformed host cellLactobacillus casei, lane 2 is the Lactobacillus casei transformantpHCE1LB:A, and lane 3 is the Lactobacillus casei transformantpHCE1LB:A-PEDN.

As described in the figures, the band corresponding to the fused proteinproduced from the expression vector pHCE1LB:A-PEDN was identified asabout 90 kDa in size.

Example 15 Analysis of Vaccine Efficacy 2 in Lactobacillus Expressing NAntigen of TGE Virus and PED Virus onto Cell Surface

The recombinant vectors pHCE1LB:A-TGEN1 and pHCE1LB:A-PEDN constructedfor surface expression in Examples 13 and 14, respectively, weretransformed into the Gram positive bacterium, Lactobacillus casei andexpression induced onto the cell surface through the same procedure asdescribed in Example 3. Thereafter, the antigenicities of the TGE virusN antigen and PED virus N antigen fused with the cell outer membraneprotein pgsA participating in the synthesis of poly-γ-glutamate weremeasured.

Essentially, the recombinant vectors pHCE1LB:A-TGEN1 and pHCE1LB:A-PEDNcreated for surface expression were transformed into Lactobacilluscasei, harvested to reach the same cell concentrations, and washedseveral times using a PBS buffer (pH 7.4). Then, 5×10 cells of theLactobacillus strain on which the N antigens had been surface-expressedwere administered orally and independently to BALB/c mice 4˜6 weeks ofage three times with a one-day interval in between, then after 1 weekinjected three times with a one-day interval in between. Four weeksafter the first administration, (1) the blood serum of the mice wascollected and the production of IgG antibodies against the N antigensexamined by performing western blotting with the N antigens(See FIG.17).

Consequently, as illustrated in FIG. 17, the blood serum from the BALB/cmice group administered the Lactobacillus transformant transformed witheither the surface expression vector pHCE1LB:A-TGEN1 (A) orpHCE1LB:A-PEDN (B) was found to include antibodies against the Nantigens.

Example 16 Construction of Recombinant Expression Vector pHCE1LB:A-PreS1and Surface Expression of PreS1 Antigen

(1) A recombinant expression vector that can express the PreS1 antigenfrom the hepatitis B virus S antigen onto a cell surface was constructedusing only the cell outer membrane protein gene pgsA from the pgsBCAgene participating in the synthesis of poly-γ-glutamate derived from aBacillus sp. strain, i.e. the surface expression vector pHCE1LB:A-PreS1,which can exploit both Gram-negative and Gram-positive bacteria as ahost cell.

To introduce the PreS1 antigenic determinant from among the hepatitis Bvirus surface antigens into the surface expression vector pHCE1LB:A, asprepared in Example 12, using Gram-positive or Gram-negative bacteria asthe host cell, about 1.5 kb of the hepatitis B virus gene was clonedinto the general cloning vector pUC8 and utilized as the template andoligonucleotides containing the nucleotide sequences of SEQ ID NO: 17and SEQ ID NO: 18 used as the primers for performing a polymerase chainreaction.

At this point, the amplified HBV S antigen gene was digested with therestriction enzymes BamHI and HindIII and ligated into the expressionvector pHCE1LB:A prepared by adjusting the translation codon at theC-terminus of the cell outer membrane protein gene pgsA, therebycreating a new recombinant expression vector, called pHCE1LB:A-PreS1, asillustrated in FIG. 18.

(2) The surface expression of the HBV PreS1 antigen was investigated inE. coli using the recombinant expression vector pHCE1LB:A-PreS1. Forthis purpose, the recombinant expression vector was transformed into E.coli and expression induced using the same procedure as described inExample 3. Then, the expression of the PreS1 antigen fused with cellouter membrane protein pgsA onto the cell surface was confirmed byperforming SDS-polyacrylamide gel electrophoresis and Westernimmunoblotting using an antibody against the PreS1 antigen (See FIG. 20in A).

As illustrated in FIG. 20, in A, lane 1 is the untransformed host cellJM109 and lane 2 is the cell transformant pHCE1LB:A-PreS1/JM109.

As a result, the band corresponding to the fused protein produced fromthe expression vector pHCE1LB:A-TGEN1 was identified to be about 55 kDain size.

Example 17 Construction of Recombinant Surface Expression VectorpHCE1LB:A-PreS2

The recombinant expression vector pHCE1LB:A-PreS2 that can express thePreS2 antigen from among the hepatitis B virus surface antigens onto acell surface was constructed using the pgsA gene from the cell outermembrane protein gene pgsBCA participating in the synthesis ofpoly-γ-glutamate derived from a Bacillus sp. strain and Gram negative orGram positive bacteria as the host cell.

To introduce the antigenic determinant region of the PreS2 antigen genesof the hepatitis B virus into the surface expression vector pHCE1LB:A,as prepared in Example 12, using Gram negative or Gram positivebacterium as the host cell, about 1.3 kb of the hepatitis B virus genewas cloned into the general cloning vector pUC8 and adopted as thetemplate and oligonucleotides with the nucleotide sequences of SEQ IDNO: 23 and SEQ ID NO: 24 utilized as the primers for performing a PCR toamplify the PreS2 antigen gene. At this point, the amplified gene regionwas about 165 bp in size. The primers with SEQ ID NO: 23 and SEQ ID NO:24 were also constructed to include the restriction enzyme BamHI andHindIII recognition sites present in the surface expression vectorpHCE1LB:A.

The PreS2 antigen gene of the HBV amplified through the polymerase chainreaction was digested with the restriction enzymes BamHI and HindIII andinserted into the C-terminal region of the cell outer membrane proteingene pgsA in the surface expression vector pHCE1LB:A already digested byadjusting the translation codon, thereby creating the new recombinantexpression vector pHCE1LB:A-PreS2, as illustrated in FIG. 18.

Example 18 Construction of Recombinant Expression VectorpHCE1LB:A-PreS1:PreS2 and Surface Expression of PreS1 Antigen

(1) A recombinant expression vector that can express a fused form of thePreS1 antigen and PreS2 antigens from among the hepatitis B virussurface antigens onto a cell surface was constructed using only the cellouter membrane protein gene pgsA from the pgsBCA gene participating inthe synthesis of poly-γ-glutamate derived from a Bacillus sp. Strain:i.e. the surface expression vector pHCE1LB:A-PreS1:PreS2, which canexploit Gram-negative or Gram-positive bacteria as the host cell.

To introduce the PreS1 and PreS2 antigenic determinants from among thehepatitis B virus surface antigens to the surface expression vectorpHCE1LB:A, as prepared in Example 12, using Gram-positive orGram-negative bacteria as the host cell, about 1.5 kb of the hepatitis Bvirus gene was cloned into the general cloning vector pUC8 and utilizedas the template and oligonucleotides containing the nucleotide sequencesof SEQ ID NO: 17 and SEQ ID NO: 24 used as the primers for performing apolymerase chain reaction. At this point, the amplified region of thegenes was 522 bp in size.

The amplified HBV PreS1:PreS2 antigen genes were digested with therestriction enzymes BamHI and HindIII and ligated into the expressionvector pHCE1LB:A prepared by adjusting the translation codon at theC-terminus of the cell outer membrane protein gene pgsA, therebycreating the new recombinant expression vector pHCE1LB:A-PreS1:PreS2, asillustrated in FIG. 19.

(2) The surface expression of the HBV PreS1 and PreS2 antigens wasinvestigated in E. coli using the recombinant expression vectorpHCE1LB:A-PreS1:PreS2. For this purpose, the recombinant expressionvector was transformed into E. coli and expression induced using thesame procedure as described in Example 3. Then, the expression of thePreS1:preS2 antigen fused with the cell outer membrane protein pgsA ontothe cell surface was confirmed by performing SDS-polyacrylamide gelelectrophoresis and Western immunoblotting using an antibody to thePreS1 antigen (See FIG. 20 in A). As illustrated in FIG. 20, in A, lane1 is the untransformed host cell JM109 and lane 3 is the celltransformant pHCE1LB:A-PreS1:PreS2/JM109.

As a result, the band corresponding to the fused protein produced fromthe expression vector pHCE1LB:A-TGEN1 was identified to be about 60 kDain size.

Example 19 Construction of Recombinant Surface Expression VectorpHCE1LB:A-L and Surface Expression of L Antigen

(1) The recombinant expression vector pHCE1LB:A-L that can express afused form of the PreS1, PreS2, and S antigens from among the hepatitisB virus surface antigens onto a cell surface was constructed using thepgsA gene from the cell outer membrane protein gene pgsBCA participatingin the synthesis of poly-γ-glutamate derived from a Bacillus sp. strainand Gram-positive or Gram-negative bacteria as the host cell. Theprimers with SEQ ID NO: 25 were also constructed to include therestriction enzyme BamHI and HindIII recognition sites present in thesurface expression vector pHCE1LB:A.

The amplified L gene of the hepatitis B virus was digested with therestriction enzymes HindIII and BamHI and ligated to the C-terminalregion of the pgsA gene from the cell outer membrane gene prepared byadjusting the translation codons. The recombinant expression vectorpHCE1LB:A-L is illustrated in FIG. 19.

(3) The surface expression of the L antigen, as a fused antigen composedof the PreS1, PreS2, and S antigens, using the surface expression vectorpHCE1LB:A-L was examined in E. coli. For this purpose, the surfaceexpression vector was transformed into E. coli host cells and expressioninduced using the same procedure as described in Example 3. Then, theexpression of the L antigen fused with the cell outer membrane proteinpgsA in the E. coli transformant was confirmed by performingSDS-polyacrylamide gel electrophoresis and Western blotting usingantibodies to the PreS1 antigen (See FIG. 20, B). As illustrated in FIG.20, lane 1 is the untransformed host cell JM109 and lanes 2 and 3 arethe cell transformant pHCE1LB:A/JM109.

As a result, the fused protein expressed from the recombinant expressionvector pHCE1LB:A-L was identified as a band of about 86 kDa.

Example 20 Construction of Recombinant Surface Expression VectorpHCE1LB:A-TNF-α

The recombinant expression vector pHCE1LB:A-TNF-α that can express afused form of the tumor necrosis factor α (TNF-α), a protein forpharmaceutical and clinical use, onto a cell surface was constructedusing the pgsA gene from the cell outer membrane protein gene pgsBCAparticipating in the synthesis of poly-γ-glutamate derived from aBacillus sp. strain and Gram-positive or Gram-negative bacteria as thehost cell.

To introduce the TNF-α gene to the surface expression vector pHCE1LB:A,as constructed in Example 12, using Gram-positive or Gram-negativebacterium as the host cell, about 0.5 kb of the TNF-α gene was clonedinto the general cloning vector pUC8 and utilized as the template andoligonucleotides containing the nucleotide sequences of SEQ ID NO: 26and SEQ ID NO: 27 used as the primers for performing a polymerase chainreaction. At this point, the amplified region of the genes was 482 bp insize. The primers with SEQ ID NO: 26 and SEQ ID NO: 27 were alsoconstructed to include the restriction enzyme BamHI and HindIIIrecognition sites present in the surface expression vector pHCE1LB:A.

The amplified L gene of the hepatitis B virus was digested with therestriction enzymes HindIII and BamHI and ligated to the C-terminalregion of the cell outer membrane gene participating in the synthesis ofpoly-γ-glutamate by adjusting the translation codons. The recombinantexpression vector pHCE1LB:A-TNF-α constructed above is depicted in FIG.21.

Example 21 Construction of Recombinant Surface Expression VectorpGNA-lipase and Surface Expression of Lipase

The recombinant expression vector pGNA-lipase that can express thelipase enzyme onto a cell surface was constructed using the pgsA genefrom the cell outer membrane protein gene pgsBCA participating in thesynthesis of poly-γ-glutamate derived from a Bacillus sp. strain and aGram-negative bacterium as the host cell.

To introduce the lipase gene to the surface expression vector pGNA usinga Gram-negative bacterium as the host cell, about 0.5 kb of the lipasegene was cloned into the general cloning vector pUC8 and utilized as thetemplate and oligonucleotides containing the nucleotide sequences of SEQID NO: 28 and SEQ ID NO: 29 used as the primers for performing apolymerase chain reaction. At this point, the amplified region of thegenes was 546 bp in size. The primers with SEQ ID NO: 28 and SEQ ID NO:29 were also constructed to include the restriction enzyme BamHI andHindIII recognition sites present in the surface expression vector pGNA.

The amplified L gene of the hepatitis B virus was digested with therestriction enzymes HindIII and BamHI and ligated to the C-terminalregion of the cell outer membrane gene participating in the synthesis ofpoly-γ-glutamate by adjusting the translation codons. The recombinantexpression vector pGNA-lipase constructed above is depicted in FIG. 22.

(2) The surface expression of the lipase gene in E. coli wasinvestigated using the recombinant expression vector pGNA-lipase. Forthis purpose, the expression vector pGNA-lipase was transformed into E.coli and expression induced through the same procedure as described inExample 3. Thereafter, the enzymatic activity of the lipase expressedonto the cell surface was assayed on an agar medium (1% Trypton, 0.5%yeast extract, 0.619% NaCl, 0.5% gum Arabic, 1 mM CaCl, 1% Tricaprylin)containing 1% of Tricaprylin with oil degradation activity. The E. colitransformant was smeared onto the agar medium containing 1% of an oilsubstrate, then cultivated in a sustained state at 37° C. for 9 hours.

As a result, the degradation of the oil substrate changed to clearregions (See FIG. 22), thereby confirming the surface expression of thelipase onto the cell surface.

Example 22 Construction of Recombinant Surface Expression VectorpGNA-amidase and Surface Expression of Amidase

(1) The recombinant expression vector pGNA-amidase that can express theamidase enzyme onto a cell surface was constructed using the pgsA genefrom the cell outer membrane protein gene pgsBCA participating in thesynthesis of poly-γ-glutamate derived from a Bacillus sp. strain and aGram-negative bacterium as the host cell.

To introduce the amidase gene to the surface expression vector pGNAusing a Gram-negative bacterium as the host cell, about 0.8 kb of theamidase gene was cloned into the E. coli expression vector pGNA andutilized as the template and oligonucleotides containing the nucleotidesequences of SEQ ID NO: 30 and SEQ ID NO: 31 used as the primers for apolymerase chain reaction. At this point, the amplified region of thegenes was 792 bp in size. The primers with SEQ ID NO: 30 and SEQ ID NO:31 were also constructed to include the restriction enzyme BamHI andHindIII recognition sites present in the surface expression vector pGNA.

The amplified L gene of the hepatitis B virus was digested with therestriction enzymes HindIII and BamHI and ligated to the surfaceexpression vector pGNA in the C-terminal region of the cell outermembrane gene participating in the synthesis of poly-γ-glutamate byadjusting the translation codons. The recombinant expression vectorpGNA-amidse constructed above is depicted in FIG. 23.

(2) The surface expression of the amidase gene in E. coli wasinvestigated using the recombinant expression vector pGNA-amidase. Forthis purpose, the expression vector pGNA-amidase was transformed into E.coli and expression induced through the same procedure as described forExample 3. Thereafter, the enzymatic activity of the amidase expressedonto the cell surface was assayed by adding the E. coli to 100 mM of aTris-HCl buffer solution (pH 8.0) containing 10 mM of D-alaNH as asubstrate and 0.5 mM of CoCl as a cofactor. In detail, the E. coli onwhich the amidase had been expressed was examined using HPLC (HypersilODS; 250×4.6 mm column) after reacting for some hours and the OD valueat 600 nm for cell growth was 1 as a unit volume. At this point, wildtype E. coli and the cell transformant containing the surface expressionvector pGNA were utilized to compare the enzymatic activities of theamidase expressed onto the cell surface.

As illustrated in FIG. 23, the E. coli transformant of the presentinvention was found to exhibit 100-fold more amidase activity than thecontrol group. Therefore, the method for surface expression in thecurrent invention was confirmed to effectively express amidase onto thecell surface.

INDUSTRIAL APPLICABILITY

As demonstrated and confirmed above, the novel expression vectors of thepresent invention can efficiently produce an exogenous protein on amicrobial surface by exploiting the cell outer membrane protein (pgsBCA)participating in the synthesis of poly-γ-glutamate derived from aBacillus sp. as a surface expression carrier and can be stably appliedto both Gram-negative and Gram-positive bacterium. The cell transformanttransformed with the surface expression vector includes an insertionsite for the targeted exogenous protein, then ligating foreign genesencoding the exogenous protein are prepared and cultivated to facilitatethe cell surface expression.

The surface expression vectors of the present invention can beeffectively used for the stable and easily detectable surface expressionof various exogenous proteins onto a cell surface regardless of the cellcycle. Consequently, the proposed microbial surface expression systemcan be utilized to produce various antigens, recombinant antibodies,recombinant enzymes, and attachment or adsorption proteins, screeningvarious antigens and antibodies, and producing enzymes for biologicalconversion. Essentially, the enzymes can be expressed onto a cellsurface and used without any reduction in the catalyst activity, therebyallowing the present invention to be industrially applied for thepurpose of bioconversion.

Those skilled in the art will appreciate that the concepts and specificembodiments disclosed in the foregoing description can be readilyutilized as a basis for modifying or designing other embodiments forcarrying out the same purposes as the current invention.

Those skilled in the art will also appreciate that such equivalentembodiments do not depart from the spirit and scope of the presentinvention as set forth in the appended claims.

1. An expression vector for producing a target protein on a microbialcell surface, which contains one or more genes selected from the groupconsisting of pgsB, pgsC and pgsA, encoding a poly-γ-glutamatesynthetase complex and a gene encoding a target protein, wherein thenucleotide sequences of pgsB, pgsC and pgsA genes comprises SEQ ID NO:1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
 2. The expressionvector for producing a target protein on a microbial cell surfaceaccording to claim 1, in which said pgsB, pgsC and pgsA genes areisolated from a Bacillus sp. strain producing poly-γ-glutamatesynthetase.
 3. The expression vector for producing a target protein on amicrobial cell surface according to claim 1, in which said gene encodingthe target protein can be selected from genes encoding enzymes,antigens, antibodies, attachment proteins, or adsorption proteins. 4.The expression vector for producing a target protein on a microbial cellsurface according to claim 1, in which said expression vector isutilized for Gram-negative bacteria.
 5. The Gram-negative bacterium thatis transformed with the expression vector for producing a target proteinon a microbial cell surface as in claim
 4. 6. The expression vector forproducing a target protein on a microbial cell surface according toclaim 1, in which said expression vector is utilized for Gram-positivebacteria.
 7. The Gram-positive bacterium that is transformed with theexpression vector for producing a target protein on a microbial cellsurface as in claim
 6. 8. The expression vector for producing a targetprotein on a microbial cell surface according to claim 1, in which saidexpression vector can be utilized for both Gram-negative andGram-positive bacteria.
 9. A method for expressing a target protein onthe microbial cell surface said method comprising: cultivating thetransformed Gram-negative bacteria of claim
 5. 10. A method forexpressing a target protein on the microbial cell surface said methodcomprising: cultivating the transformed Gram-positive bacteria of claim7.
 11. An expression vector for producing a target protein on amicrobial cell surface, which contains one or more genes selected fromthe group consisting of pgsB, pgsC and pgsA, encoding a poly-γ-glutamatesynthetase complex and a gene encoding a target protein, wherein saidpgsB, pgsC and pgsA genes are isolated from a Bacillus sp. strainproducing poly-γ-glutamate synthetase.